Identification and Characterization of Functionally Important Elements in the Multidrug Resistance Protein 1 COOH-terminal Region

Abstract

The ATP binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), transports a broad spectrum of conjugated and unconjugated compounds, including natural product chemotherapeutic agents. In this study, we have investigated the importance of the COOH-terminal region of MRP1 for transport activity and basolateral plasma membrane trafficking. The COOH-terminal regions of some ABCC proteins have been implicated in protein trafficking, but the function of this region of MRP1 has not been defined. In contrast to results obtained with other ABCC proteins, we found that the COOH-proximal 30 amino acids of MRP1 can be removed without affecting trafficking to basolateral membranes. However, the truncated protein is inactive. Furthermore, removal of as few as 4 COOHterminal amino acids profoundly decreases transport activity. Although amino acid sequence conservation of the COOH-terminal regions of ABC proteins is low, secondary structure predictions indicate that they consist of a broadly conserved helix-sheet-sheet-helix-helix structure. Consistent with a conservation of secondary and tertiary structure, MRP1 hybrids containing the COOH-terminal regions of either the homologous MRP2 or the distantly related P-glycoprotein were fully active and trafficked normally. Using mutated proteins, we have identified structural elements containing five conserved hydrophobic amino acids that are required for activity. We show that these are important for binding and hydrolysis of ATP by nucleotide binding domain 2. Based on crystal structures of several ABC proteins, we suggest that the conserved amino acids may stabilize a helical bundle formed by the COOH-terminal three helices and may contribute to interactions between the COOH-terminal region and the protein's two nucleotide binding domains.

Role of the NH2-terminal Membrane Spanning Domain of Multidrug Resistance Protein 1/ABCC1 in Protein Processing and Trafficking

“…The results obtained with MRP1 204 -1531 -YFP suggested that elements that contributed to targeting of the MRP1 core to the plasma membrane might be located in the COOH-terminal region of the protein. However, we found recently that up to 30 amino acids could be removed from the COOH terminus of full-length MRP1 without affecting trafficking, despite the fact that removal of only four amino acids was sufficient to markedly decrease transport activity (Westlake et al, 2004). As observed previously, subcellular localization of MRP1 1-1501 and MRP1 1-1527 was indistinguishable from that of the full-length protein ( Figure 6, A and B).…”

“…In contrast, processing and trafficking of SUR1A and MRP2 is impaired after removal of only the COOH-terminal seven and 15 amino acids, respectively (Sharma et al, 1999;Nies et al, 2002). The different effects of COOH-terminal truncation of MRP1 and MRP2 was unexpected because substitution of COOH-terminal region of MRP1 with that of MRP2 had no effect on trafficking (Konno et al, 2003;Westlake et al, 2004), even in the absence of MSD0 (Figure 8A), suggesting a high level of functional conservation between the COOH-terminal regions of the two MRPs.…”

“…Processing and Trafficking of MRP1 Hybrid Proteins Recently, it has been shown that the COOH-terminal region following the Walker B motif in NBD2 of MRP1 can be exchanged for the analogous domain from MRP2 without affecting activity or plasma membrane localization (Konno et al, 2003;Westlake et al, 2004). Consequently, we determined whether this was also true in the absence of MSD0.…”

“…In contrast, proteins lacking MSD0, as well as the COOH-terminal four or 30 amino acids, failed to exit the ER (Figure 6, C and D). We also have shown that a double Ala substitution of the highly conserved dileucine motif in the COOH-terminal region of MRP1, Leu 1514 -1515 has no effect on trafficking of the full-length protein (Westlake et al, 2004). However, in the absence of MSD0, this mutation resulted in extensive colocalization of the MRP1 core with ER marker calnexin ( Figure 6E), whereas more conservative substitution of the dileucine motif with Val or Ile did not ( Figure 6F and Supplementary Data Figure 3A).…”

“…MRP1 expression vectors encoding proteins lacking the first 203 amino acid residues or with mutated/chimeric COOH-terminal regions have been described previously (Grant et al, 1994;Gao et al, 1996;Qian et al, 2001a;Westlake et al, 2003Westlake et al, , 2004. Vectors for proteins that contained both modified NH 2 -and COOH-terminal regions were generated by ligation of mutant MRP1 fragments from the above-mentioned vectors into an appropriate recipient vector.…”